With the completion of mapping the human genome, science has ushered in a new frontier of research and development. Scientists are now undertaking studies to understand the underlying proteins which make up the genetic code. Proteomics is the study of proteins which make up the twenty-three common amino acids that can be created from the nucleic acid bases of genomics. Proteomics can be much more complex than genomics due to the extremely large number of proteins that can be “spelled” by the twenty-three amino acids. Further, photometric assays are usually quantitative, providing information about the concentration of particular proteins, not just their mere presence or absence.
Two common macroscopic methods for purifying proteins are currently used. First, gel electrophoresis is a technique that involves electric-field induced migration of the proteins through gels. Electrophoresis can be inexpensive and is generally achieves high purity. However, it is slow since separation is based on diffusion of the proteins through a gel medium.
A second common method for protein identification is matrix-assisted laser desorption ionization (MALDI). MALDI is a mass spectroscopic, time-of-flight method based on laser desorption of bio-molecules in a vacuum chamber. While sensitive and capable of high purity, specific (due to many fragments of different masses from a given protein) and potentially quantitative, MALDI is a slow process and requires expensive, non-portable equipment.
Many new proteomic screening methods based on antibody-antigen binding have been developed using lithographic technology to design assays built into microchips. The microchips can contain devices designed to be coated with complementary antibodies which allow a specific protein to bind to the device.
These new assays have the advantage of being relatively fast and inexpensive, but they are applicable to generalized testing given their reliance on the availability of complimentary antibodies. In other words, if the proper binding antibody is not provided by the assays, the presence or absence of a protein requiring that particular binding antibody will remain unknown. When complimentary antibodies are not available, non-specific binding to the device may occur, but such binding is by no means a true indicator for identification.
Existing micro-cantilever based on-chip assays rely on optical detection of a static cantilever deflection when a protein unfolds. A laser, LED, or other optical source can be used to measure the amount of deflection, which can then be used to determine the specific types of proteins. When complex biological fluids, such as for example blood, are used rather than test solutions like buffered saline, optical opacity may be a serious problem and the entire analysis and identification process may be thwarted.
On-chip detection, identification and quantization of proteins in complex solutions such as blood are highly desirable for a large number of health monitoring and screening applications. For example, rapid home tests for HIV invention or pathogens in food could save countless lives. Portability of the test equipment for home use and field use beyond the confines of a laboratory is also highly desirable so as to expedite the identification of pathogens and identify the source so as to limit further exposure.
On-chip detection methods can be faster and cheaper than those involving high-vacuum systems or lasers. However, most on-chip tests identify proteins through observation of specific antibody binding reactions. When on-chip detectors utilize non-specific binding mechanisms, accuracy is lost as they lose the ability to identify specific proteins.
Microcantilever devices provide nanomechanical motion in response to thermal change. The microcantilevers have a high surface area to volume ratio, which permits detection of surface stresses that are too small for observation on a macroscale. The microcantilever devices are used in a wide variety of physical, chemical, and biological sensing applications, as reported by Lavrik et al., Cantilever transducers as a platform for chemical and biological sensors, 74 Review of Scientific Instruments, 2229 (2004).
The mode of action is to convert changes in Gibbs free energy into a mechanical response, for example, as reported by Hagen et al., Nanomechanical Forces Generated by Surface Grafted DNA, 106 J. Phys. Chem 10163 (2002). Analyte-adsorbate interactions and adsorbate-adsorbate interactions are known to induce mechanical responses, as reported by Fritz et al., Translating Biomolecular Recognition into Nanomechanics, 338 Science 316 (2000). A common type of microcantilever device is a silicon nitride beam, for example, of 200 μm thickness, with a gold layer deposited to perhaps ten percent of this thickness.
The transduction of a chemical signal into a mechanical response may occur according to Stoney's formula, which predicts a bending moment in the microcantilever device in response to surface stress. Microcalorimeter devices are available on commercial order, for example, from MicroCal, LLC of Northhampton, Md.
One type of microcalorimetry application is isothermal titration calorimetry. In this type of system, a syringe is used to inject an analyte, and an adiabatic shield surrounds two cells. One cell is a reference cell and the other is a sample cell that is positioned to receive the analyte. The two cells are maintained at a temperature differential where the reference cell is maintained at constant power and the sample cell is maintained by using power that is proportional to a temperature difference between the sample cell and the reference cell. The calorimetric behavior of the analyte is assessed as a difference between a calibration run (without the analyte) and a separate run with the analyte being present.
The ability to detect extremely small thermal changes provides a platform for investigation into chemical changes that were previously unquantifiable. In various examples, isothermal titration calorimetry has been used to study protein interactions including those for small molecules—enzyme inhibition, protein-carbohydrate, protein-protein, protein-lipid, protein-nucleic acid, protein folding, and protein stability. Isothermal titration calorimetry has also been used to study nucleic acids including nucleic acid-small molecule interactions, nucleic acid-nucleic acid interactions, and nucleic acid melting. Isothermal titration calorimetry has also been used to study antibodies, cell receptors, enzymes, lipid interactions, non-biological interactions, and other reactions.
Some prior system use lasers to monitor cantilever deflection. The use of laser energy for this purpose is problematic because it tends to provide heating action that is a source of noise in the measurements. Accordingly, it is problematic to adapt microcantilever equipment for purposes of differential scanning calorimetry.
Hence there is a need for a system that overcomes one or more of the drawbacks identified above.